1. Field of the Invention
The present invention relates to purified signal peptides for use in facilitating secretion of one or more proteins from plant cells. The present invention also relates to nectarins, proteins derived from nectar which possess oxalate oxidase activity. The present invention also relates to methods for treating diseases or conditions related to abnormal oxalate deposition.
2. Description of the Background
Genetically engineered proteins are expressed in two ways: via intracellular expression and via secreted expression. In intracellular expression, host cells are isolated and homogenized to form an extract from which expressed proteins are purified. Extract production involves mechanical disruption of the host cells, followed by extraction of soluble proteins. The ease of host cell extraction varies by cell type. Animal cells can easily be homogenized with a hand homogenizer, but yeast and bacteria require vigorous mechanical treatment. Plant cells are particularly difficult to disrupt because they have durable cell walls. Also, plant cells have subcellular organelles containing compounds such as proteases, polyphenols, and alkaloids which, upon disruption, rapidly degrade and inactivate proteins. Furthermore, cell extracts may comprise over one thousand (1000) different species of proteins. Depending on the intended use of the expressed proteins, substantial effort may be required for their isolation and purification from such complex environments.
Secreted expression, on the other hand, lacks many of these drawbacks. Secreted proteins are easier to purify because the extracellular environment into which proteins are secreted, has few, if any, contaminating proteins. With the attachment of a signal peptide, a host cell can be directed to secrete most proteins of interest.
In the natural state, signal peptides guide proteins from their synthesis site to their functional location. Secretory signal peptides guide proteins outside the cell, and are typically about fifteen to about sixty amino acids long. After a protein is properly transported, the signal peptide is removed and has no further effect on the function of the mature protein.
The mechanism of protein secretion has been reviewed by Davis and Tai, Nature 283, 433-438 (1980). In all species, the exact amino acid sequence of the signal peptide appears to be less important than the physical properties of the amino acids that constitute the peptide. For example, an acidic amino acid may be replaced with another acidic amino acid without impairing signal peptide function.
Plants, like other organisms, utilize signal sequences to direct protein transport. These signal sequences, like their mammalian counterparts, are interchangeable and functionally independent of the attached protein. Shieh et al., Plant Physiol. 101, 353-361 (1993). When proteins non-native or foreign to a plant cell are attached to a signal peptide, they are transported and processed in a similar fashion to endogenous or native plant cell proteins.
Few systems have been developed for the production of secreted proteins. E. coli usually does not produce secreted proteins because of its simplicity, and yeasts secrete only small proteins. While mammalian cells readily secrete proteins, the secreted proteins are difficult to purify because they are often expressed at low levels. Plant secretory signal peptides are known to exist because some plant proteins are known to occur extracellularly. However, plant secretory peptides have heretofor not been isolated. Consequently, methods using signal peptides for secreted expression of proteins from plants have not been developed.
Plants and plant cells have unique advantages as host organisms for protein production. Plant cultivation and plant cell culture is less demanding than mammalian cell culture. A number of attributes of plants and plant cells contribute to their low maintenance cost. For example, plants and plant cells do not require expensive media and do not generate liquid biohazardous waste. The ability of plants and plant cells to grow in a wide range of temperature, humidity, and oxygen levels also contributes to their low maintenance cost. Additionally, proteins produced from bacteria or other micro-organisms are not adequately glycosylated. In contrast, plants and plant cells glycosylate proteins using the same molecular machinery as animals.
Consequently, there is a strong need for a method which provides for protein secretion into plant secretory materials. The ability to isolate and purify proteins from plant secretory materials would allow an efficient and cost-effective means of using plants to express recombinant proteins.
Disclosed herein are proteins identified in the secretory material of plant nectar. The proteins, called nectarins, are secreted via mechanisms which utilize signal peptides. As will become apparent from the following detailed description, these and other signal peptides can be used to transport recombinant proteins into secretory material. Furthermore, it has been discovered that the nectarins possess oxalate oxidase activity. The importance of this finding will now be addressed.
Diseases or conditions associated with abnormal deposition or regulation of oxalate take a strong clinical toll. For example, urolithiasis (kidney stones) is responsible for seven to ten of every one thousand hospital admissions in the United States. Also, disorders such as hereditary primary oxaluria and secondary oxaluria associated with enteric disease leads to abnormal deposition of oxalate. The most popular method for treating kidney stones is lithotripsy. However, this method is dependent upon identification of stones after they have already been formed. Diet is often manipulated to reduce the risk of developing kidney stones. About 75% of stones are composed of calcium oxalate or calcium oxalate mixed with calcium phosphate. A pharmaceutical agent possessing the ability to catalyze the degradation of oxalate would therefore have significant clinical utility for treating diseases or conditions related to abnormal deposition of oxalate. Additionally, methods for determining oxalate concentrations in fluids and biological specimens often utilize coupled enzymatic reacting employing oxalate oxidase. Proteins possessing oxalate oxidase activity therefore have utility in these and other detection methods.